Coumarines useful as biomarkers

ABSTRACT

The invention provides compounds of formula (I), wherein R, R 1 , R 2 , R 3  and R 4  are as defined in the description, and their preparation. The compounds of formula I are useful as biomarkers.

This application claims the benefit under U.S.C. § 365 (a) ofinternational application No. PCT/EP 03/02251, filed Mar. 5, 2003, whichin turn claims the benefit under U.S.C. § 365 (b) and U.S.C. § 119(a) offoreign application GB 0205281.9, filed Mar. 6, 2002.

The present invention relates to novel coumarine derivatives, theirpreparation, their use as markers and compositions containing them.

More particularly the invention provides a compound of formula I

wherein

-   either R₁ and R₂ are both hydrogen and either R₃ and R₄,    independently, are H, CH₃, ¹¹CH₃, (CH₂)_(n)I, (CH₂)_(n) ¹²³I,    (CH₂)_(n)OH, (CH₂)_(n)F or (CH₂)_(n) ¹⁸F, n being 2, 3 or 4, or R₃    and R₄, together with the nitrogen atom to which they are attached,    form a group of formula

-    wherein R₅ is H, (CH₂)_(n)I, (CH₂)_(n) ¹²³I, (CH₂)_(n)OH, CH₃,    ¹¹CH₃, (CH₂)_(n)F or (CH₂)_(n) ¹⁸ F, n being as defined above,-   or one of R₁ and R₂ is hydrogen and the other, together with R₃,    forms a —(CH₂)_(m)—bridge, m being 2 or 3, and R₄ is H, CH₃,    (CH₂)_(n)I, (CH₂)_(n) ¹²³I, (CH₂)_(n)OH, ¹¹CH₃, (CH₂)_(n)F or    (CH₂)_(n) ¹⁸F, and-   R is a group of formula

-    wherein X is O, S or NR₈, R₈ being H, CH₃, ¹¹CH₃, (CH₂)_(n)I,    (CH₂)_(n) ¹²³I, (CH₂)_(n)OH, (CH₂)_(n)F or (CH₂)_(n) ¹⁸F (n being as    defined above), Y is CH or N and R₆ and R₇, independently, are H,    NO₂, F, ¹⁸F, O(CH₂)_(n)F, O(CH₂)_(n) ¹⁸F, Cl, CN, ¹¹CN, OCH₃,    O¹¹CH₃, I, ¹²³I, O(CH₂)_(n)I or O(CH₂)_(n) ¹²³I (n being as defined    above),    in free base or acid addition salt form, for use as a marker.

The compounds of formula I, with the exception of the followingcompounds:

-   7-Dimethylamino-3-(1-methyl-1H-benzoimidazol-2-yl)-chromen-2-one-   3-(1H-Benzoimidazol-2-yl)-7-dimethylamino-chromen-2-one-   3-(6-Chloro-benzothiazol-2-yl)-7-dimethylamino-chromen-2-one-   3-Benzothiazol-2-yl-7-dimethylamino-chromen-2-one-   3-Benzooxazol-2-yl-7-dimethylamino-chromen-2-one-   3-Benzooxazol-2-yl-7-methylamino-chromen-2-one-   3-(5-Chloro-benzooxazol-2-yl)-7-dimethylamino-chromen-2-one    have never been disclosed in the literature and are part of the    present invention.

In a further aspect, the invention provides a process for the productionof the compounds of formula I and their salts, comprising the steps of

-   a) for the production of a compound of formula I wherein R₃, R₄, R₅,    R₆, R₇ and R₈ are different from ¹¹CH₃, (CH₂)_(n) ¹⁸F, (CH₂)_(n)    ¹²³I, ¹⁸F, O(CH₂)_(n) ¹⁸F, ¹¹CN, O¹¹CH₃, ¹²³I and O(CH₂)_(n) ¹²³I,    reacting a compound of formula II with a compound of formula III

-   -   wherein R₃ and R₄ as well as R₅ in R₃ and R₄; R₆ and R₇ in R;        and R₈ in X are different from ¹¹CH₃, (CH₂)_(n) ¹⁸F, (CH₂)_(n)        ¹²³I, ¹⁸F, O(CH₂)_(n) ¹⁸F, ¹¹CN, O¹¹CH₃, ¹²³I and O(CH₂)_(n)        ¹²³I, and Alk is (C₁₋₄)alkyl, or

-   b) for the production of a compound of formula I wherein at least    one of R₆ and R₇ is O¹¹CH₃, reacting a compound of formula I wherein    at least one of R₈ and R₇ is OH with I¹¹CH₃ and a base, or

-   c) for the production of a compound of formula I wherein at least    one of R₆ and R₇ is O(CH₂)_(n) ¹⁸F, respectively O(CH₂)_(n) ¹²³I,    reacting a compound of formula I wherein at least one of R₆ and R₇    is O(CH₂)_(n)OTs or O(CH₂)_(n)OMs with ¹⁸F^(Θ), respectively    ¹²³I^(Θ), or

-   d) for the production of a compound of formula I wherein at least    one of R₆ and R₇ is ¹⁸F, reacting a compound of formula I wherein at    least one of R₆ and R₇ is NO₂ or halogen, with ¹⁸F^(Θ), or

-   e) for the production of a compound of formula I wherein at least    one of R₆ and R₇ is ¹²³I, reacting a compound of formula I wherein    at least one of R₆ and R₇ is Bu₃Sn, with ¹²³I and hydrogen peroxide,    or

-   f) for the production of a compound of formula I wherein at least    one of R₆ and R₇ is ¹¹CN, reacting a compound of formula I wherein    at least one of R₆ and R₇ is OSO₂CF₃ with [¹¹C]cyanide, or

-   g) for the production of a compound of formula I wherein at least    one of R₃, R₄, R₅ and R₈ is ¹¹CH₃, reacting a compound of formula I    wherein at least one of R₃, R₄, R₅ and R₈ is hydrogen, with ¹¹CH₃I,    or

-   h) for the production of a compound of formula I wherein at least    one of R₃, R₄, R₅ and R₈ is (CH₂)_(n) ¹⁸F, respectively (CH₂)_(n)    ¹²³I, reacting a compound of formula I wherein at least one of R₃,    R₄, R₅ and R₈ is (CH₂)_(n)OTs or (CH₂)_(n)OMs with ¹⁸F^(Θ),    respectively I^(Θ),    and recovering the resulting compound of formula I in free base form    or in form of an acid addition salt.

The reactions can be effected according to known methods, for example asdescribed in the Examples.

Working up the reaction mixtures and purification of the compounds thusobtained may be carried out in accordance to known procedures.

Acid addition salts may be produced from the free bases in known manner,and vice-versa.

Compounds of formula I In free base or acid addition salt form,hereinafter referred to as agents of the invention, exhibit valuableproperties as histopathological staining agents, imaging agents and/orbiomarkers, hereinafter “markers”.

More particularly the agents of the invention are useful as markers forlabeling pathological structures such as intraneuronal neurofibrillarytangles and extracellular β-amyloid plaques, e.g. in the brain ofpatients with Alzheimer's disease (see Example 5).

The agents of the invention are therefore useful for the early diagnosisand prevention of Alzheimer's disease and for monitoring theeffectiveness of therapeutic treatments of Alzheimer's disease.

The advantages of assessing amyloid and neurofibril deposition in vivoand non-invasively using markers capable of labeling these structureshave been reported e.g. in WO 00/10614.

In accordance with the above, the present invention provides acomposition for labeling histopathological structures in vivo or invitro, comprising an agent of the invention.

In a further aspect, the present invention provides a method forlabeling histopathological structures in vitro or in vivo, whichcomprises contacting brain tissue with an agent of the invention.

Said brain tissue comprises for example B-amyloid plaques and/orneurofibrillary tangles.

Contacting the brain tissue with the agent of the invention is forexample effected by administering the agent of the invention to apatient, e.g. a patient with Alzheimer's disease.

The method of the invention may comprise a further step aimed atdetermining whether the agent of the invention labeled the targetstructure.

If the agent of the invention is a non-radioactive compound of formulaI, said further step may be effected by observing the target structureusing fluorescence microscopy.

If the agent of the invention is a radioactive compound of formula I,said further step may be effected by observing the target structureusing positron emission tomography (PET) or single photon emissioncomputed tomography (SPECT).

Labeling histopathological structures in vitro is effected, for example,for detecting histopathological hallmarks of Alzheimer's disease.

Labeling histopathological structures in vivo is effected, for example,for diagnosing Alzheimer's disease in a patient or for monitoring theeffectiveness of a therapeutic treatment of Alzheimer's disease.

The following examples illustrate the invention.

EXAMPLE 13-Benzothiazol-2-yl-7-[4-(2-fluoro-ethyl)-piperazin-1-yl]-chromen-2-one

200 mg (0.793 mmol)4-[4-(2-Fluoro-ethyl)-piperazin-1-yl]-2-hydroxy-benzaldehyde and 164 mg(1 eq.) benzothiazol-2-yl-acetic acid methyl ester are heated to refluxfor 3 h in 5 mL benzene and 2.5 mL acetonitrile, in the presence of0.157 mL (2 eq.) piperidine. The reaction mixture is allowed to reachroom temperature and the precipitate filtered off, washed withdiethylether and dried under high vacuum to yield 90 mg (55%) of desiredproduct as a yellow powder (melting point: 246° C.).

¹H-NMR (400 MHz, CDCl₃): δ=8.95 (s, 1H); 8.03, 7.97 (2d, 2H); 7.53 (D,1H); 7.50, 7.39 (2t, 2H); 6.88 (dd,1H); 6.78 (d,1H); 4.62 (dt, 2H); 3.45(t, 4H); 2.78 (dt, 2H); 2.70 (t, 4H).

The starting materials are prepared as described hereafter:

3-[4-(2-Fluoro-ethyl)-piperazin-1 -yl]-phenol

1 g (5.61 mmol) 3-Piperazin-1-yl-phenol and 0.5 mL (1.25 eq)1-bromo-2-fluoroethane are stirred 20 h at 60° C. in 5 mL DMF, thereaction mixture allowed to reach room temperature and evaporated, andthe residue column chromatographed (silica gel, ethyl acetate/petroleumether 9:1) to yield 600 mg (48%) of the desired product as a brown oil.

4-[4-(2-Fluoro-ethyl)piperazin-1 -yl]-2-hydroxy-benzaldehyde

600 mg (2.675 mmol) 3-[4-(2-Fluoro-ethyl)-piperazin-1-yl]-phenol aredissolved in 8 mL DMF and cooled to 0° C. 0.27 mL (1.1 eq) POCl₃ isadded dropwise within 2 min. and the reaction mixture stirred for anadditional 5 min. before being allowed to reach room temperature, andthen heated and stirred for 3 h at 90° C. The reaction mixture isevaporated and the residue extracted with water and ethyl acetate. Theorganic phases are washed with brine, dried over sodium sulphate andevaporated. The residue is column chromatographed (silica gel, ethylacetate followed by ethyl acetate/MeOH 85:15) to yield the desiredproduct as a yellowish oil.

Benzothiazol-2-yl-acetic acid methyl ester

3.2 mL (30 mmol) 2-Amino-thiophenol are dissolved in 100 mL diethyletherand treated with 4.17 mL (1 eq.) triethylamine to yield a suspension, towhich 3.21 mL (1 eq.) chlorocarbonyl-acetic methyl ester in 10 mLdiethylether is added dropwise within 20 min. The resulting suspensionis stirred and additional 2 h at room temperature, and the precipitateremoved by filtration. The filtrate is evaporated and columnchromatographed (silica gel, ethyl acetate/petroleum ether 1:2) to yieldthe desired product as a yellowish liquid (5.1 g, 82%).

Alternatively,3-Benzothiazol-2-yl-7-[4-(2-fluoro-ethyl)piperazin-1-yl]-chromen-2-onecan be prepared as described hereafter:

10 mg (0.025 mmol)3-benzothiazol-2-yl-7-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-chromen-2-oneare dissolved in 3 mL dichloromethane with 2.5 mg (0.8 eq.)4-dimethylaminopyridine and 0.013 mL (3 eq.) diisopropylethylamine, andcooled to 0° C. 5.7 mg (1.2 eq.) tosyl chloride are added and thereaction mixture stirred for one hour before being allowed to reach roomtemperature. After an additional two hours stirring, the reactionmixture is evaporated, the residue taken up in tetrahydrofurane andtreated with 0.2 mL tetrabutylammonium fluoride (1M in THF). Afterstirring for 30 minutes, the solution is evaporated and the desiredproduct obtained as a yellow powder.

MS(EI+): 410 (M+1).

The preparation of the starting material is described in Example 3.

EXAMPLE 23-(6-Chloro-imidazo[1,2-a]pyridin-2-yl)-8-(2-fluoro-ethyl)-5,6,7,8-tetrahydro-1-oxa-8-aza-anthracen-2-one

200 mg (0.896 mmol)1-(2-Fluoro-ethyl)-7-hydroxy-1,2,3,4-tetrahydro-quinoline-6-carbaldehydeand 201 mg (1 eq.) (6-chloro-imidazo[1,2-a]pyridin-2-yl)-acetic acidmethyl ester in 6 mL benzene and 3 mL acetonitrile are treated with0.177 mL (2 eq.) piperidine and refluxed for 18 h. The reaction mixtureis allowed to reach room temperature and the precipitate filtered,washed with acetonitrile and dried under high vacuum to yield 240 mg(67%) of desired product as a yellow powder.

¹H-NMR (400 MHz, CDCl₃): δ=8.59, 8.44,8.16 (3s, 3H); 7.51, 7.15 (2d,2H); 7.13 (s, 1H); 4.68, 3.68 (2dt, 2CH₂); 3.50, 2.82 (2dt, 2CH₂); 1.99(m, CH₂).

MS(EI+): 398 (M+1).

M.p.=290° C. (decomposition).

The starting materials are prepared as described hereafter.

1 -(2-Fluoro-ethyl)7-hydroxy-1,2,3,4-tetrahydro-quinoline-6-carbaldehyde

290 mg (1.48 mmol)1-(2-Fluoro-ethyl)-7-hydroxy-1,2,3,4-tetrahydro-quinolin-7-ol aredissolved in 5 mL DMF. 0.15 mL POCl₃ are added dropwise at 0° C. Thereaction mixture is slowly heated to 50° C., stirred an additional 3 hat this temperature, then cooled to RT and extracted with ethyl acetateand an aqueous saturated solution of sodium bicarbonate. The combinedorganic phases are washed with brine, dried over sodium sulphate andevaporated. The residue is column chromatographed (silica gel, ethylacetate/petroleum ether 1:2) to yield 200 mg (60%) desired product as abrown solid.

1-(2-Fluoro-ethyl)-1,2,3,4-tetrahydro-quinolin-7-ol

500 mg (3.35 mmol) 1,2,3,4-Tetrahydro-quinolin-7-ol are dissolved in 8mL DMF and stirred at 60° C. overnight with 0.325 mL (1.3 eq)1-bromo-2-fluoroethane, in the presence of 0.63 mL (1.1 eq)diisopropylethylamine. The reaction mixture is extracted with 0.1 N aqu.HCl and ethyl acetate. The combined organic extracts are washed withbrine, dried over sodium acetate and evaporated. The residue is columnchromatographed (silica gel, ethyl acetate/petroleum ether 1:2 to 1:1)to yield 290 mg (44%) desired product as a brown oil.

(6-Chloro-imidazo[1,2-a]pyridin-2-yl)-acetic acid methyl ester

1.28 g (10 mmol) 2-Amino-5-chloropyridine and 1.18 mL (1 eq)4-chloro-3-oxo-butyric acid methyl ester are heated to 115° C. in 15 mLtoluene for 18 h. The resulting brown suspension is evaporated andheated 1 h to 90° C. under high vacuum, then cooled to room temperatureand stirred in 100 mL dichloromethane, 10 mL saturated aqueous sodiumbicarbonate solution and 40 mL water at 0° C. for one hour. The organicphase is then separated and evaporated to yield a brownish solid that iscolumn chromatographed (silica gel, ethyl acetate/petroleum ether 4:1)to yield 700 mg (31 %) desired product as a beige powder. MS(EI+):225-227 (M+1).

EXAMPLE 33-Benzothiazol-2-yl-7-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-chromen-2-one

240 mg crude 3-benzothiazol-2-yl-7-piperazin-1-yl-chromen-2-one in 10 mLDMF are stirred for 72 h at room temperature in the presence of 858 mg(at least 4 eq.) cesium carbonate and 0.103 mL (at least 2 eq.)2-iodoethanol. The reaction mixture is extracted with ethyl acetate anda saturated solution of sodium carbonate and washed with brine. Thecombined organic phases are dried with sodium sulphate and evaporated.The residue is column chromatographed (silica gel,dichloromethane/methanol 95:5+1% conc. NH₄OH) to yield 70 mg (26%)desired product as an orange solid.

¹H-NMR (400 MHz, CDCl₃): δ=8.97 (s, 1H); 8.03, 7.97 (2d, 2H); 7.56 (1d,1H); 7.50, 7.39 (2t, 2H); 6.89 (d, 1H): 8.78 (s,1H); 3.70 (m, CH₂);3.47, 2.72 (2m, 2×2CH₂); 2.64 (m, CH₂).

MS(EI+): 408 (M+1).

The starting material is prepared as described hereafter:

3-Benzothiazol-2-yl-7-piperazin-1-yl-chromen-2-one

890 mg (5 mmol) 3-Piperazin-1-yl-phenol are suspended in 100 mL EtOH andtreated with 0.42 mL (1 eq.) 12N HCl at a temperature below 10° C. Thesuspension is stirred an hour and slowly evaporated. The remaining beigepowder is dried under high vacuum, then taken up in 60 mL DMF. Thesolution is cooled to 5° C. under argon, 0.503 mL (1.1 eq.) POCl₃ addeddropwise and the reaction mixture stirred for an hour at roomtemperature, then 30 minutes at 80° C., and finally allowed to cool toroom temperature, upon which 2 g (3 eq.) solid potassium carbonate isadded. The suspension is stirred 30 minutes at room temperature thenevaporated, and the residue resuspended and stirred in a 1:1 mixture ofdichloromethane and isopropanol. The suspension is filtered, thefiltrate evaporated and dried to yield 600 mg crude2-hydroxy4-piperazin-1-yl-benzaldehyde. This material is taken up in 40mL of a 1:1 mixture of benzene and acetonitrile, 2.5 mL (5 eq.)piperidine and 683 mg (0.66 eq.) benzothiazol-2-yl-acetic acid methylester are added under argon and the reaction mixture refluxed for anhour. The product is extracted with dichloromethane and brine at 0° C.,the combined organic phases dried with sodium sulphate, filtered andevaporated, then triturated with diisopropylether to yield 800 mgdesired product (crude).

EXAMPLE 43-(6-Chloro-imidazo[1,2-a]pyridin-2-yl)-7-[4-(2-fluoro-ethyl)-piperazin-1-yl]-chromen-2-one

253 mg (1 mmol)4-[4-(2-Fluoro-ethyl)-piperazin-1-yl]-2-hydroxy-benzaldehyde and 450 mg(2 eq.) (6-chloro-imidazo[1,2-a]pyridin-2-yl)-acetic acid methyl esterrefluxed in 15 mL benzene and 7.5 mL acetonitrile in the presence of0.395 mL (4 eq.) piperidine for 21 h. The reaction mixture is cooled to0° C. and the precipitated material filtered, washed with diethyl etherand dried under high vacuum, yielding 100 mg (23%) desired product as ayellow solid. M.p.=265° C. (decomposition).

EXAMPLE 5 3-Benzothiazol-2-yl-7-(4-methyl-piperazin-1-yl)-chromen-2-one

100 mg (0.28 mmol) 3-Benzothiazol-2-yl-7-piperazin-1-yl-chromen-2-onewere dissolved in 15 mL DMF and treated under argon with 183 mg (2 eq.)cesium carbonate, cooled below 5° C. and treated with 1 eq (0.018 mL)Mel. After stirring for 4 h, the reaction mixture is diluted with 100 mLdichloromethane and washed twice with ice-water. The organic phase isevaporated and the residue column chromatographed (silica gel,dichloromethane/EtOH 9:1). Recrystallization frommethanol/dichloromethane yields 50 mg (47%) desired product as an orangepowder.

MS(EI+): 378 (M+1).

EXAMPLE 63-Benzothiazol-2-yl-7-[(2-fluoro-ethyl)-methyl-amino]-chromen-2-one

130 mg (ca. 0.65 mmol) crude4-[(2-Fluoro-ethyl)-methyl-amino]-2-hydroxy-benzaldehyde and 202 mg (1.5eq.) benzothiazol-2-yl-acetic acid methyl ester are dissolved in 3 mLacetonitrile and 6 mL benzene, treated with 0.14 mL (2 eq.) piperidineand heated to reflux for 45 minutes. After cooling to room temperature,the crude product is extracted with AcOEt/isopropanol 9:1 and saline,dried over sodium sulfate and evaporated. The crude product is dissolvedin 20 mL methanol, and slowly concentrated down to 10 mL at 50° C. Theorange crystals are filtered and dried under high vacuum to yield 110 mg(48%) desired product.

M.p.=231-233° C.

The starting materials are prepared as described hereafter:

4-[(2-Fluoro-ethyl)-methyl-amino]-2-hydroxy-benzaldehyde

169 mg (1 mmol) 3-[(2-Fluoro-ethyl)-methyl-amino]-phenol are dissolvedin 4 mL DMF and cooled below 10° C. 0.101 mL (1.1 eq.) POCl₃ are addeddropwise over the course of one minute, the reaction mixture is stirred10 minutes at room temperature, then heated to 90° C. for 30 minutes.The reaction mixture is allowed to cool to 30° C. and cautiously treatedwith a slow addition of 8 mL saturated aqueous sodium bicarbonatesolution. The desired product is extracted with ethyl acetate andsaline, the organic phase dried over sodium sulfate and evaporated toyield 130 mg (66%) crude product as a slightly brownish oil. Thiscompound is used without further purification.

MS(EI+): 198 (M+1).

3-[(2-Fluoro-ethyl)methyl-amino]-phenol

450 mg (2.46 mmol) (2-Fluoro-ethyl)-(3-methoxy-phenylymethyl-amine aredissolved in 1 mL acetic acid and treated with 5 mL of a 33% solution ofHBr in acetic acid for 20 h, at 100° C. After cooling to roomtemperature, the reaction mixture is poured onto ice, treated with 8 mLof a 4N aqueous NaOH solution and extracted with ethyl acetate. Theorganic phase is washed with saline and an aqueous solution of sodiumbicarbonate, then evaporated to yield 400 mg of a brownish oil. Thecrude product is column chromatographed (silica gel, ethylacetate/petroleum ether 1:4) to yield 320 mg (77%) of desired product asa brownish resin.

MS(EI+): 170 (M+1).

(2-Fluoro-ethyl)-(3-methoxy-phenyl)-methyl-amine

350 mg (ca. 1 mmol) crude toluene-4-sulfonic acid2-[(3-methoxy-phenyl)-methyl-amino]-ethyl ester are dissolved in 10 mLTHF under argon and treated with 4 mL (4 eq.) of a 1N solution ofn-tetrabutylammonium fluoride in THF at 70° C. for 2 h. The reactionmixture is cooled to room temperature and poured onto ice, thenextracted with TBME. The organic phase is washed with saline and anaqueous solution of sodium bicarbonate, dried over sodium sulfate andevaporated to yield 180 mg (99%) desired product as a brownish oil. Thiscompound is used without further purification.

MS(EI+): 184 (M+1).

Toluene4-sulfonic acid 2-[(3-methoxy-phenyl)-methyl-amino]-ethyl ester

181 mg (1 mmol) 2-[(3-methoxy-phenyl)-methyl-amino]-ethanol is dissolvedin 8 mL dichloromethane and after addition of 0.2 mL (2.5 eq.) pyridinecooled below 5° C. A solution of 489 mg (1.5 eq) p-toluenesulfonic acidanhydride in 4 mL dichloromethane is added dropwise and the reactionmixture stirred at room temperature for 30 minutes before ice and 100 mLTBME are added, followed by 70 mL of a cold aqueous solution of sodiumbicarbonate. The organic phase is separated, washed with saline, thendried over sodium sulfate and evaporated. The residue is taken up intoluene and evaporated to yield 350 mg (quant.) crude desired product asa brownish oil. This compound is used without further purification.

¹H-NMR (400 MHz, CDCl₃): δ=7.70, 7.25 (2d, 2×2H); 7.04 (t, H); 6.24,6.19 (2d, 2H); 6.11 (br. S, H); 6.89 (d, 1H); 4.17 (t, CH₂); 3.78 (s,Me); 3.59 (t, CH₂); 2.86, 2.41 (2s, 2Me).

EXAMPLE 77-[(2-Fluoro-ethyl)-methyl-amino]-3-(5-thiophene-2-yl-[1,3,4]oxadiazol-2-yl)-chromen-2-one

40 mg (ca. 0.2 mmol) crude4-[(2-Fluoro-ethyl)-methyl-amino]-2-hydroxy-benzaldehyde and 30 mg (0.13mmol) (5-thiophen-2-yl-[1,3,4]oxadiazol-2-yl)-acetic acid methyl esterare dissolved under argon in 4 mL benzene and 2 mL acetonitrile, treatedwith 0.03 mL (2 eq.) piperidine and heated to reflux for 1 h. Thereaction mixture is allowed to reach room temperature, evaporated, andthe residue column chromatographed (silica gel, AcOEt/petroleum ether1:1). The fluorescent, yellow product is triturated in diisopropylether,then dried under high vacuum to yield 9 mg (19%) desired product as ayellow powder.

MS(EI+): 372 (M+1).

The starting material (5-thiophen-2-yl-[1,3,4]oxadiazol-2-yl)-aceticacid methyl ester is prepared in a similar manner to the known(5-thiophen-2-yl-[1,3,4]oxadiazol-2-yl)-acetic acid ethyl ester(Heterocycl Commun 2001 7(5):411-416).

EXAMPLE 83-(4-Buta-1,3-dienyl-5-methyl-thiazol-2-yl)-7-[4-(3-fluoro-propyl)-piperazin-1yl]-chromen-2-one

50 mg (0.087 mmol) Toluene-4-sulfonic acid3-[4-(3-benzothiazol-2-yl-2oxo-2H-chromen-7-yl)-piperazin-1-yl]-propylester are dissolved in 10 mL anhydrous THF, treated with 0.18 mL of a 1Nsolution of n-tetrabutylammonium fluoride in THF and heated to 60° C.for 2 h. The reaction mixture is poured onto ice and extracted withdichloromethane after addition of 2 mL aqueous sodium bicarbonatesolution. The organic phase is evaporated and the residue columnchromatographed (silica gel, dichloromethane/EtOH 98:2) to yield afterevaporation and drying under high vacuum 9 mg (26%) desired product as alight brownish powder.

The starting materials are prepared as described hereafter:

Toluene-4-sulfonic acid3-[4-(3-benzothiazol-2-yl-2-oxo-2H-chromen-7-yl)-piperazin-1-yl]-propylester

70 mg (0.16 mmol)3-Benzothiazol-2-yl-7-[4-(3-hydroxy-propyl)-piperazin-1-yl]-chromen-2-oneare dissolved in 15 mL dichloromethane under argon. 0.083 mL Hünig baseand 10 mg DMAP are added to the stirred solution, followed by 46 mg (1.5eq.) tosyl chloride. The reaction mixture is stirred for 20 h at roomtemperature, poured onto ice, treated with 2 mL aqueous sodiumbicarbonate solution and extracted (dichloromethane, saline). Theorganic phase is evaporated and the residue column chromatographed(silica gel, dichloromethane/EtOH 98:2) to yield after evaporation anddrying under high vacuum 73 mg (78%) desired product as a light brownishpowder.

MS(EI+): 576 (M+1).

3-Benzothiazol-2-yl-7-[4-(3-hydroxy-propyl)-piperazin-1-yl]-chromen-2-one

320 mg (1 mmol)3-{4-[3-(tetrahydro-pyran-2-yloxy)-propyl]-piperazin-1-yl}-phenol isdissolved in 10 mL DMF and treated below 10° C. with 0.1 mL POCl₃. Thereaction mixture is stirred at room temperature for 6 hours, thenevaporated and the crude product taken up in 10 mL acetonitrile andtreated with 0.6 mL (6 eq.) piperidine. 207 mg benzothiazol-2-yl-aceticacid methyl ester in 10 mL toluene are added and the reaction mixturestirred for 30 minutes at 90° C. before being evaporated. The residue istaken up in 20 mL anhydrous THF, stirred for two hours in the presenceof 1 mL of a 6N aqueous HCl solution and evaporated. The residue istaken up in dichloromethane and washed with aqueous sodium bicarbonateand saline. The organic phase is evaporated and the residue columnchromatographed (silica gel, dichloromethane/EtOH/aq. NH₃ 90:9.9:0.1) toyield 70 mg (17%) desired product as a yellow-orange powder.

MS(EI+): 422 (M+1).

3-{4-[3-(Tetrahydro-pyran-2-yloxy)-propyl]-piperazin-1 -yl}-phenol

712 mg (4 mmol) 3-Piperazin-1-yl-phenol are dissolved in 30 mL DMF underargon and cooled below 5° C. After addition of 1.38 g (2.5 eq.) K₂CO₃,166 mg (0.25 eq.) KI and 888 mg (1 eq.)2-(3-bromo-propoxy)-tetrahydro-pyran, the suspension is stirred at roomtemperature for 20 hours, then extracted with AcOEt after dilution withsaline. The organic phase is dried over sodium sulfate, evaporated andthe residue column chromatographed (silica gel, AcOEt/EtOH 95:5) toyield 630 mg (48%) desired product as a light yellowish resin.

MS(EI−): 319 (M−1).

EXAMPLE 9 3-Benzooxazol-2-yl-7-(4-methyl-piperazin-1-yl)-chromen-2-one

240 mg (0.79 mmol)7-(4-Methyl-piperazin-1-yl)-2-oxo-2H-chromene-3-carboxylic acid aredissolved in 12 mL MeOH and heated to 50° C. for three hours in thepresence of 2.36 mL KOH in MeOH/water 9:1. After cooling to roomtemperature, the reaction mixture is acidified with a 6N aqueous HCLsolution to pH 2, evaporated and dried under high vacuum to yield ayellow powder. This product is reacted without further purificationafter dissolution in 10 mL diglyme and 10 mL acetonitrile with 0.115 mL(2 eq.) thionyl chloride and 0.1 mL DMF. The reaction mixture is heatedto 70° C. for 90 minutes, cooled to room temperature, concentrated to avolume of 10 mL, and treated with 105 mg (1.2 eq.) 2-Amino-phenol and400 mg polyphosphoric acid before stirring for 2 h at 170° C. The cooledreaction mixture is extracted with dichloromethane in the presence of anaqueous solution of sodium carbonate, and the organic phase evaporatedto yield the amide as a crude product. This compound is dissolved in 10mL DMF, treated with 0.1 mL POCl₃ and heated to 60° C. for three hours.The reaction mixture is cooled to room temperature, treated with anaqueous solution of sodium bicarbonate and extracted withdichloromethane. The organic phase is evaporated and columnchromatographed (silica gel, dichloromethane/EtOH/aq. ammonia90:9.9:0.1). The product is recrystallized from dichloromethane/methanoland dried under high vacuum to yield 20 mg desired product asorange-yellow crystals (7% overall).

MS(EI+): 362 (M+1).

¹H-NMR (400 MHz, CDCl₃): δ=8.63 (s, 1H), 7.81, 7.59, 7.48, 7.36, 6.84(5m, 6H); 6.73 (s, 1H); 3.43, 2.58 (2m, 2×2CH₂); 2.38 (s, Me).

The starting materials are prepared as described hereafter:

7-(4-Methyl-piperazin-1 -yl)-2oxo-2H-chromene-3-carboxylic acid

546 mg (2 mmol) 3-(4-Methyl-piperazin-1-yl)-phenol hydrobromide aredissolved in 15 mL DMF and treated with 0.2 mL (1.1 eq.) POCl₃, at atemperature below 5° C. After heating to 50° C. and stirring for 20hours, the reaction mixture is cooled to room temperature and treatedwith water. After stirring for an additional two hours, the product isextracted with ethyl acetate in the presence of an aqueous solution ofsodium bicarbonate, and the organic phase washed twice with water, driedover sodium sulfate and evaporated to yield 580 mg of a light brownresin which is further used without additional purification.

The intermediate product is dissolved in 10 mL MeOH, treated with 0.23mL (1 eq.) malonic acid dimethyl ester and 0.02 mL piperidine, refluxedfor one hour, cooled to room temperature and evaporated. The residue iscolumn chromatographed (silica gel, dichloromethane/EtOH/aq. ammonia90:9.9:0.1) to yield 240 mg (39% overall) desired product as a yellowsolid.

MS(EI+): 303 (M+1).

EXAMPLE 10

Staining of APP23 Mouse and Human Alzheimer Disease (AD) Brain Sectionsusing an Agent of the Invention or Thioflavine S.

Four-micrometer thick paraffin sections from an APP23 mouse at 26 monthsof age are deparaffinized in xylene and rehydrated. 10 mg of thecompound are dissolved in 1 ml DMSO and diluted with deionized water1:10. This staining solution is applied on sections for about 20 min.Section background is cleared by washing with 95% ethanol. Finallysections are dehydrated in 99% ethanol, cleared in xylene and mountedwith Vectashield™. Sections are investigated using fluorescencemicroscopy with the following filter combination: Excitation 450-490 nm,emission 510 nm. Twenty micrometer thick dryotom sections from a ADbrain cortex are air dried and fixated in 4% PFA for 5 min. Afterwashing in tap water sections are stained either with Thioflavine S orwith the compound for 5 min and further processed as described above.The compound is dissolved in DMSO and diluted to a final concentrationof 0.01% with 50% Ethanol, Thioflavine S is dissolved in 50% Ethanol,final concentration is 0.01%.

In vivo Labeling of Aβ in APP23 Mice with the Agent of the Invention

Injection solution is prepared fresh by dissolving 10 mg of the compoundin 0.2 mL DMSO diluted with 9.8 ml sterile water. Lower concentrationsare prepared by further dilution with water. Four APP23 female mice at21 month of age receive one single injection of the compound (Injectionvolume: 1 ml/100 gr body weight). The treated animals are killed bydecapitation after one hour. The brains are removed and frozen on dryice. 14 μm thick sections are cut in a cryotome, thawmounted andair-dried. Staining is performed as described above. Sections areanalyzed using conventional fluorescence microscopy and confocalmicroscopy.

Results

-   1) Staining of APP23 mice brain sections (which contain amyloid    deposits but no neurofibrillary tangles):

The agents of the invention strongly stain amyloid plaques and vascularamyloid deposits in brain sections of APP23 mice.

-   2) Staining of human AD brain sections (which contain both amyloid    deposits and neurofibrillary tangles):

Brain sections taken from frontal cortex of AD patients are stained withthe agents of the invention, and the results compared with a ThioflavineS stain. The agents of the invention intensely and selectively stainamyloid deposits and neuroribrillary tangles.

-   3) Ex vivo staining in APP23 mice:

Intravenous administration of the agents of the invention in APP23 miceleads to a selective and intense staining of amyloid deposits, analyzedex vivo.

1. A compound of the formula

in which R₁ and R₂ are both hydrogen; R₃ and R₄, together with thenitrogen atom to which they are attached, form a group of the formula

in which R₅ is hydrogen, (CH₂)_(n)I, (CH₂)_(n) ¹²³I, (CH₂)_(n)OH, CH₃,¹¹CH₃, (CH₂)_(n)F or (CH₂)_(n) ¹⁸F, n being 2, 3, or 4; and R is a groupof the formula

in which X is S, Y is N, and R₆ and R₇, independently, are hydrogen,NO₂, F, ¹⁸F, O(CH₂)_(n)F, O(CH₂)_(n) ¹⁸F, Cl, CN, ¹¹CN, OCH₃, O¹¹CH₃, I,¹²³I, O(CH₂)_(n)I or O(CH₂)_(n) ¹²³I, n being 2, 3, or 4; in free baseform or in acid addition salt form.
 2. The compound according to claim 1of the formula I, which is3-benzothiazol-2-yl-7-[4-(2-fluoro-ethyl)-piperazin-1-yl]-chromen-2-one,in free base form or in acid addition salt form.
 3. A composition forlabeling histopathological structures in vitro or in vivo, comprising acompound as defined in claim 1 of the formula I, in free base form or inacid addition salt form.
 4. A method for labeling histopathologicalstructures in vitro or in vivo, comprising contacting brain tissue witha compound as defined in claim 1 of the formula I, in free base form orin acid addition salt form.